• The CD3 complex (comprising CD3ε, CD3δ, CD3γ，and CD3ζ subunits) is a critical transmembrane signaling module of the T-cell receptor (TCR). These subunits form heterodimers that non-covalently associate with TCRαβ to mediate immune synapse formation and signal transduction.
• BCMA (TNFRSF17), a member of the tumor necrosis factor receptor superfamily (TNFRSF), is a transmembrane glycoprotein pivotal for plasma cell survival and immune regulation. Expressed predominantly on terminally differentiated B cells and malignant plasma cells, BCMA binds APRIL (TNFSF13) and BAFF (TNFSF13B) to activate NF-κB and PI3K/AKT pathways, driving cell proliferation and drug resistance in multiple myeloma (MM).
• GPRC5D, an orphan receptor in the class C GPCR family, is selectively expressed on plasma cells and malignant cells in multiple myeloma (MM). GPRC5D is overexpressed in MM patients resistant to BCMA-targeted therapies, positioning it as a novel immunotherapeutic target.
• Chimeric human CD3EDG were expressed, while mouse Cd3edg were knocked out in B-hCD3EDG/hBCMA/hGPRC5D mice.
• The exon 1 and part of exon 2 of the mouse Tnfrsf17 gene, which encode part of the extracellular domain, were replaced by human counterparts in B-hCD3EDG/hBCMA/hGPRC5D mice. The genomic region of mouse Tnfrsf17 gene that encodes transmembrane domain and cytoplasmic portion were retained. The promoter, 5’UTR and 3’UTR region of the mouse gene were also retained. The human TNFRSF17 expression was driven by endogenous mouse Tnfrsf17 promoter, while mouse Tnfrsf17 gene transcription and translation will be disrupted.
• The exons 1-3 of mouse Gprc5d gene that encode the whole molecule (ATG to STOP codon) were replaced by human counterparts in B-hCD3EDG/hBCMA/hGPRC5D mice. The promoter, 5’UTR and 3’UTR region of the mouse gene were retained. The human GPRC5D expression was driven by endogenous mouse Gprc5d promoter, while mouse Gprc5d gene transcription and translation will be disrupted.

Strain specific analysis of CD3E, CD3D and CD3G mRNA expression in wild-type C57BL/6 mice and B-hCD3EDG/hBCMA/hGPRC5D mice by RT-PCR. Spleen RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hCD3EDG/hBCMA/hGPRC5D mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human CD3E, CD3D and CD3G primers. Mouse Cd3e, Cd3d and Cd3g mRNA was only detectable in wild-type C57BL/6 mice. Human CD3E, CD3D and CD3G mRNA was detectable in homozygous B-hCD3EDG/hBCMA/hGPRC5D mice but not in wild-type mice.

Strain specific analysis of BCMA and GPRC5D mRNA expression in wild-type C57BL/6 mice and B-hCD3EDG/hBCMA/hGPRC5D mice by RT-PCR. Spleen RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hCD3EDG/hBCMA/hGPRC5D mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human BCMA and GPRC5D primers. Mouse Bcma and Gprc5d mRNA was detectable in wild-type mice. Human BCMA and GPRC5D mRNA was detectable in homozygous B-hCD3EDG/hBCMA/hGPRC5D mice but not in wild-type mice.

CD3E expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCD3EDG/hBCMA/hGPRC5D mice by flow cytometry. Spleen T cells were collected from wild-type C57BL/6 mice (+/+) (male, 7-week-old, n=1) and homozygous B-hCD3EDG/hBCMA/hGPRC5D mice (H/H; H/H; H/H; H/H; H/H) (male, 7-week-old, n=1). Protein expression was analyzed with anti-human CD3E antibody (BD Horizon™, 562426) and anti-mouse CD3E antibody (Biolegend, 100312) by flow cytometry. Human CD3E was exclusively detectable in homozygous B-hCD3EDG/hBCMA/hGPRC5D mice, but not in wild-type C57BL/6 mice.

CD3E expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCD3EDG/hBCMA/hGPRC5D mice by flow cytometry. Blood T cells were collected from wild-type C57BL/6 mice (+/+) (male, 7-week-old, n=1) and homozygous B-hCD3EDG/hBCMA/hGPRC5D mice (H/H; H/H; H/H; H/H; H/H) (male, 7-week-old, n=1). Protein expression was analyzed with anti-human CD3E antibody (BD Horizon™, 562426) and anti-mouse CD3E antibody (Biolegend, 100312) by flow cytometry. Human CD3E was exclusively detectable in homozygous B-hCD3EDG/hBCMA/hGPRC5D mice, but not in wild-type C57BL/6 mice.

BCMA expression analysis in wild-type C57BL/6 mice and homozygous B-hCD3EDG/hBCMA/hGPRC5D mice by flow cytometry. Spleen plasma cells were collected from wild-type C57BL/6 mice and homozygous B-hCD3EDG/hBCMA/hGPRC5D mice. Protein expression was analyzed with human BCMA antibody (Biolegend, 357504). Human BCMA was detectable in spleen of B-hCD3EDG/hBCMA/hGPRC5D mice but not in wild-type C57BL/6 mice.

Note: Both groups of mice were treated using an undisclosed methods.

GPRC5D expression analysis in wild-type C57BL/6 mice and homozygous B-hCD3EDG/hBCMA/hGPRC5D mice by flow cytometry. Spleen plasma cells were collected from wild-type C57BL/6 mice (male, 7-week-old) and homozygous B-hCD3EDG/hBCMA/hGPRC5D mice (male, 7-week-old). Protein expression was analyzed with an GPRC5D targeting antibody (in house). GPRC5D was detectable in spleen from both wild-type C57BL/6 mice and B-hCD3EDG/hBCMA/hGPRC5D mice, attributable to confirmed cross-reactivity of the antibody with the mouse GPRC5D, as previously validated in tumor cell lines (data not shown).